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1.
Int J Cancer ; 145(4): 1020-1032, 2019 08 15.
Article in English | MEDLINE | ID: mdl-30873613

ABSTRACT

Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor, is a polyomavirus-induced human cancer. To study the causal relationship of MCC carcinogenesis with the integrated Merkel cell polyomavirus (MCPyV) in detail, well-characterized MCC cell lines are needed. Consequently, in the current study, we established and characterized six MCPyV-positive MCC cell lines. Microarray-based comparative genomic hybridization revealed a stable genome carrying only a limited number of chromosomal gains and deletions. All cell lines expressed MCC markers Keratin-20 and neuron-specific enolase as well as truncated MCPyV-encoded large T antigen (LT). For five cell lines, we were able to identify the MCPyV-integration sites in introns of different genes. The LT-truncating stop codon mutations and integration sites were affirmed in the respective clinical patient samples. Inverse PCR suggested that three of the cell lines contained MCPyV genomes as concatemers. This notion was confirmed for the two cell lines with known integration sites. Importantly, our observation of distinct stop codon mutations in cell lines with concatemeric MCPyV integration indicates that these LT-truncating mutations occur before integration. In summary, we provide the detailed characterization of six MCPyV-positive MCC cell lines, which are likely to serve as valuable tools in future MCC research.


Subject(s)
Antigens, Viral, Tumor/genetics , Carcinoma, Merkel Cell/genetics , Merkel cell polyomavirus/genetics , Polyomavirus Infections/genetics , Tumor Virus Infections/genetics , Animals , Carcinoma, Merkel Cell/virology , Cell Line, Tumor , Codon, Terminator/genetics , Genome, Viral/genetics , Humans , Mice , Mutation/genetics , Polyomavirus Infections/virology , Skin Neoplasms/genetics , Skin Neoplasms/virology , Tumor Virus Infections/virology
2.
Cancer Immunol Immunother ; 68(6): 983-990, 2019 Jun.
Article in English | MEDLINE | ID: mdl-30993371

ABSTRACT

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer in which PD-1/PD-L1 blockade has shown remarkable response rates. However, a significant proportion of patients shows primary or secondary resistance against PD-1/PD-L1 inhibition, with HLA class-I downregulation and insufficient influx of CD8+ T cells into the tumor as possible immune escape mechanisms. Histone deacetylase inhibitors (HDACi) have been demonstrated to reverse low HLA class-I expression caused by epigenetic downregulation of the antigen machinery (APM) in vitro and in pre-clinical models in vivo. CASE PRESENTATIONS: We report four cases of patients with metastatic MCC who did not respond to immunotherapy by PD-1/PD-L1 blockade. Two of the patients received, subsequently, the HDACi panobinostat in combination with PD-1/PD-L1 blockade. Tumor biopsies of the patients were analyzed for cellular and molecular markers of antigen processing and presentation as well as the degree of T-cell infiltration. RESULTS AND CONCLUSION: Low expression of APM-related genes associated with low HLA class-I surface expression was observed in all MCC patients, progressing on PD-1/PD-L1 blockade. In one evaluable patient, of the two treated with the combination therapy of the HDACi, panobinostat and PD-1/PD-L1 blockade, reintroduction of HLA class-I-related genes, enhanced HLA class-I surface expression, and elevated CD8+ T-cell infiltration into the MCC tumor tissue were observed; however, these changes did not translate into a clinical benefit. Our findings suggest that HDACi may be useful to overcome HLA class-I downregulation as a resistance mechanism against anti-PD-1/PD-L1 antibodies in MCC patients. Prospective clinical trials are needed to evaluate this notion.


Subject(s)
Antibodies, Monoclonal/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , Carcinoma, Merkel Cell/drug therapy , Histocompatibility Antigens Class I/immunology , Histone Deacetylase Inhibitors/therapeutic use , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Skin Neoplasms/drug therapy , Aged , Aged, 80 and over , Antibodies, Monoclonal/immunology , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/immunology , Down-Regulation/drug effects , Down-Regulation/genetics , Down-Regulation/immunology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Histone Deacetylase Inhibitors/immunology , Humans , Immunotherapy/methods , Male , Middle Aged , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , Skin Neoplasms/genetics , Skin Neoplasms/immunology
3.
Cancer Immunol Immunother ; 67(3): 341-351, 2018 03.
Article in English | MEDLINE | ID: mdl-29188306

ABSTRACT

Merkel cell carcinoma (MCC) is a highly aggressive, often lethal neuroendocrine cancer. Its carcinogenesis may be either caused by the clonal integration of the Merkel cell polyomavirus into the host genome or by UV-induced mutations. Notably, virally-encoded oncoproteins and UV-induced mutations affect comparable signaling pathways such as RB restriction of cell cycle progression or p53 inactivation. Despite its low incidence, MCC recently received much attention based on its exquisite immunogenicity and the resulting major success of immune modulating therapies. Here, we summarize current knowledge on epidemiology, biology and therapy of MCC as conclusion of the project 'Immune Modulating strategies for treatment of Merkel Cell Carcinoma', which was funded over a 5-year period by the European Commission to investigate innovative immunotherapies for MCC.


Subject(s)
Carcinoma, Merkel Cell/epidemiology , Carcinoma, Merkel Cell/therapy , Skin Neoplasms/epidemiology , Skin Neoplasms/therapy , Animals , Carcinoma, Merkel Cell/virology , Europe , Humans , Immunotherapy/methods , Merkel cell polyomavirus/pathogenicity , Skin Neoplasms/virology
4.
J Dtsch Dermatol Ges ; 15(9): 887-893, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28872233

ABSTRACT

The identification of targetable molecules in cellular signaling pathways represents a milestone in the treatment of melanoma. Selective inhibitors of these molecules, known as phosphokinases, allow for individual signaling pathways to be "switched off". This is of particular importance for tumors in which these pathways are constitutively activated by mutations in genes encoding said molecules. Especially patients with BRAF-mutated melanomas significantly benefit from kinase inhibitor therapies, with the current standard of combined BRAF and MEK inhibition providing very good long-term disease control. Such regimens have been shown to achieve a progression-free survival of more than ten months and an overall survival of more than two years, along with good quality of life. Given that the majority of patients develop secondary resistance during long-term kinase inhibitor therapy, current clinical trials are geared towards finding suitable drug combinations including inhibitors of other signaling pathways as well as immune checkpoint inhibitors. The present review highlights targeted therapies for melanoma currently available as well as potential future options presently under clinical investigation.


Subject(s)
Melanoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Skin Neoplasms/drug therapy , Clinical Trials as Topic , DNA Mutational Analysis , Disease-Free Survival , Drug Therapy, Combination , GTP Phosphohydrolases/antagonists & inhibitors , GTP Phosphohydrolases/genetics , Humans , Melanoma/genetics , Melanoma/mortality , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Treatment Outcome
5.
J Dtsch Dermatol Ges ; 15(9): 887-894, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28872247

ABSTRACT

Die Behandlungsstrategie beim metastasierten Melanom hat sich mit der Identifizierung therapeutisch angreifbarer molekularer Zielstrukturen innerhalb zellulärer Signalwege radikal geändert. Durch die Zulassung von Substanzen, die gezielt an den zentralen Schaltmolekülen, den Phosphokinasen, angreifen, können diese Signalwege selektiv abgeschaltet werden. Dies ist insbesondere bei denjenigen Tumoren von Interesse, deren Signalwege durch aktivierende Mutationen der für die Schaltmoleküle kodierenden Gene konstitutiv aktiviert sind. Aktuell ist diese therapeutische Strategie insbesondere für Patienten bedeutsam, deren Melanome eine Mutation im BRAF-Gen aufweisen. Diese Patienten können durch eine Kombinationstherapie aus Inhibitoren der Phosphokinasen BRAF und MEK langfristig mit sehr guter Krankheitskontrolle behandelt werden. Unter dieser Kombinationstherapie wird aktuell ein progressionsfreies Überleben von über zehn Monaten und ein Gesamtüberleben von mehr als zwei Jahren bei guter Lebensqualität erzielt. Da unter längerfristiger Therapie mit Kinaseinhibitoren jedoch bei einem Großteil der Patienten eine Resistenzbildung auftritt, sind aktuelle klinische Therapiestudien auf die Suche nach geeigneten Kombinationspartnern unter Blockierung anderer Signalwege oder unter Aktivierung der T-Zell-vermittelten Immunantwort ausgerichtet. Der vorliegende Übersichtsartikel stellt sowohl die aktuell verfügbaren als auch die in der klinischen Testung befindlichen zukünftigen Optionen der zielgerichteten Therapie des Melanoms dar.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Melanoma/drug therapy , Molecular Targeted Therapy , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effects , Skin Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , Melanoma/enzymology , Melanoma/genetics , Protein Kinase Inhibitors/adverse effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Signal Transduction/genetics , Skin Neoplasms/enzymology , Skin Neoplasms/genetics
6.
Cancer Immunol Immunother ; 60(2): 227-34, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20981424

ABSTRACT

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.


Subject(s)
Antibodies/blood , Antibodies/immunology , Cyclin B1/immunology , Epitopes, T-Lymphocyte/immunology , Health , Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Adult , Aged , Aged, 80 and over , Algorithms , Cells, Cultured , Cyclin B1/chemistry , Enzyme-Linked Immunosorbent Assay , HLA-A2 Antigen/immunology , Humans , Middle Aged , Neoplasms/blood
7.
Oncogene ; 40(5): 980-996, 2021 02.
Article in English | MEDLINE | ID: mdl-33311552

ABSTRACT

Merkel cell carcinoma (MCC) is a highly invasive and metastatic skin cancer. While high expression of miR-375 is a characteristic of MCC, it seems not to contribute to the malignant phenotype of MCC cells. miR-375 enrichment in MCC-derived extracellular vesicles suggests its intercellular signaling function. Here, we demonstrate that horizontally transferred miR-375 causes fibroblast polarization toward cancer-associated fibroblasts (CAFs). The polarization is evidenced by phenotypic changes and induction of α-SMA, CXCL2, and IL-1ß. Fibroblast polarization is inhibited by specific antagomirs and mimicked by experimental miR-375 expression. Mechanistically, miR-375 downregulates RBPJ and p53, two key players regulating fibroblast polarization. In clinical MCC samples, in situ hybridization located miR-375 in CAFs, which correlated with high α-SMA protein and low RBPJ and TP53 expression; single-cell RNAseq revealed a disparate fibroblast polarization negatively correlating with p53 pathway-related gene expression. Thus, the functional role of miR-375 in MCC is to generate a pro-tumorigenic microenvironment by inducing fibroblast polarization.


Subject(s)
Carcinoma, Merkel Cell/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/antagonists & inhibitors , MicroRNAs/genetics , Tumor Suppressor Protein p53/antagonists & inhibitors , Actins/genetics , Antagomirs/pharmacology , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Carcinogenesis/genetics , Carcinoma, Merkel Cell/pathology , Cell Polarity/genetics , Chemokine CXCL2/genetics , Exosomes/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Interleukin-1beta/genetics , RNA-Seq , Signal Transduction/genetics , Single-Cell Analysis , Tumor Microenvironment/genetics , Tumor Suppressor Protein p53/genetics
8.
J Invest Dermatol ; 141(7): 1675-1686.e4, 2021 07.
Article in English | MEDLINE | ID: mdl-33600825

ABSTRACT

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer characterized by high invasiveness, early metastases, and high mortality. Because of the lack of suitable animal models, most functional studies are performed using cell lines, some of which lack classical neuroendocrine growth characteristics. Here, we scrutinized the molecular characteristics of classical MCC and variant MCC cell lines by differential gene expression and the respective epigenetic regulation by microRNAs and DNA methylation. Cutaneous squamous cell carcinoma cell lines were used for comparison. The most striking observation was a lower expression of epithelial-mesenchymal transition-related genes in classical MCCs, which was accompanied by higher expression of the epithelial-mesenchymal transition-regulating microRNA clusters miR-200c-141 and miR-183-96-182 and hypomethylation of the respective microRNA loci. Experimental expression of the MCC lineage factor ATOH1 in variant MCCs resulted in an increased expression of miR-200c-141 paralleled by a reduction of genes associated with epithelial-mesenchymal transition, thus demonstrating a connection between neuroendocrine characteristics and the lack of epithelial-mesenchymal transition. Together, our observations not only reinforce concerns about the use of variant MCCs as proper MCC representatives, but also suggest variant MCCs as cells locked in an intermediate state between neuroendocrine and epithelial differentiation.


Subject(s)
Carcinoma, Merkel Cell/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Skin Neoplasms/genetics , Carcinoma, Merkel Cell/pathology , Carcinoma, Squamous Cell/pathology , Cell Differentiation/genetics , Cell Line, Tumor , DNA Methylation , Epigenesis, Genetic , Epithelial-Mesenchymal Transition/genetics , Humans , Skin/pathology , Skin Neoplasms/pathology
9.
J Invest Dermatol ; 140(1): 56-65.e3, 2020 01.
Article in English | MEDLINE | ID: mdl-31283928

ABSTRACT

Despite the fact that the transcription factor ATOH1 is a master regulator of Merkel cell development, its role in Merkel cell carcinoma (MCC) carcinogenesis remains controversial. Here, we provide several lines of evidence that ATOH1 is a lineage-dependent oncogene in MCC. Luciferase assays revealed binding of ATOH1 and subsequent activation to the promoter of miR-375, which is one of the most abundant microRNAs in MCCs. Overexpression of ATOH1 in variant MCC cell lines and fibroblasts induced miR-375 expression, whereas ATOH1 knockdown in classical MCC cell lines reduced miR-375 expression. Moreover, ATOH1 overexpression in these cells changed their growth characteristics from adherent to suspension and/orspheroidal growth, that is, resembling the neuroendocrine growth pattern of classical MCC cell lines. Notably, ectopic expression of different Merkel cell polyomavirus (MCPyV)-derived truncated large T antigens induced ATOH1 expression in fibroblasts, which was paralleled by miR-375 expression and similar morphologic changes. In summary, MCPyV-associated carcinogenesis is likely to induce the characteristic neuroendocrine features of MCC via induction of ATOH1; thus, ATOH1 can be regarded as a lineage-dependent oncogene in MCC.


Subject(s)
Antigens, Viral, Tumor/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Merkel Cell/genetics , Merkel cell polyomavirus/physiology , MicroRNAs/genetics , Oncogenes/genetics , Skin Neoplasms/genetics , Antigens, Viral, Tumor/metabolism , Carcinogenesis , Cell Differentiation , Cell Line, Tumor , Cell Lineage , Gene Expression Regulation, Neoplastic , Humans , Polyomavirus Infections , Tumor Virus Infections
10.
Clin Cancer Res ; 26(9): 2257-2267, 2020 05 01.
Article in English | MEDLINE | ID: mdl-31932494

ABSTRACT

PURPOSE: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, which can be effectively controlled by immunotherapy with PD-1/PD-L1 checkpoint inhibitors. However, a significant proportion of patients are characterized by primary therapy resistance. Predictive biomarkers for response to immunotherapy are lacking. EXPERIMENTAL DESIGN: We applied Bayesian inference analyses on 41 patients with MCC testing various clinical and biomolecular characteristics to predict treatment response. Further, we performed a comprehensive analysis of tumor tissue-based immunologic parameters including multiplexed immunofluorescence for T-cell activation and differentiation markers, expression of immune-related genes and T-cell receptor (TCR) repertoire analyses in 18 patients, seven objective responders, and 11 nonresponders. RESULTS: Bayesian inference analyses demonstrated that among currently discussed biomarkers only unimpaired overall performance status and absence of immunosuppression were associated with response to therapy. However, in responders, a predominance of central memory T cells and expression of genes associated with lymphocyte attraction and activation was evident. In addition, TCR repertoire usage of tumor-infiltrating lymphocytes (TILs) demonstrated low T-cell clonality, but high TCR diversity in responding patients. In nonresponders, terminally differentiated effector T cells with a constrained TCR repertoire prevailed. Sequential analyses of tumor tissue obtained during immunotherapy revealed a more pronounced and diverse clonal expansion of TILs in responders indicating an impaired proliferative capacity among TILs of nonresponders upon checkpoint blockade. CONCLUSIONS: Our explorative study identified new tumor tissue-based molecular characteristics associated with response to anti-PD-1/PD-L1 therapy in MCC. These observations warrant further investigations in larger patient cohorts to confirm their potential value as predictive markers.


Subject(s)
B7-H1 Antigen/antagonists & inhibitors , Carcinoma, Merkel Cell/pathology , Immune Checkpoint Inhibitors/therapeutic use , Immunologic Memory , Lymphocytes, Tumor-Infiltrating/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptors, Antigen, T-Cell/genetics , Aged , Bayes Theorem , Biomarkers, Tumor/analysis , Carcinoma, Merkel Cell/drug therapy , Carcinoma, Merkel Cell/immunology , Carcinoma, Merkel Cell/metabolism , Female , Humans , Male , Skin Neoplasms/drug therapy , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Treatment Outcome
11.
J Clin Invest ; 130(8): 4266-4281, 2020 08 03.
Article in English | MEDLINE | ID: mdl-32427578

ABSTRACT

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases before immune checkpoint blockade (ICB) correlated with nonresponsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription, thereby overcoming T cell resistance. Antigen presentation was restored in interferon-sensitive (IFN-sensitive) but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-Ihi (DDX58hi) melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the antitumor T cell activity of ICB nonresponders. Overall, the herein-identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , DEAD Box Protein 58/immunology , Gene Silencing , Immunity, Cellular , Immunotherapy , Melanoma, Experimental/immunology , Neoplasm Proteins/immunology , Animals , CD8-Positive T-Lymphocytes/pathology , Cell Line, Tumor , DEAD Box Protein 58/genetics , Humans , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/genetics , Receptors, Immunologic , Xenograft Model Antitumor Assays
12.
Cancer Immunol Res ; 7(9): 1472-1484, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31266785

ABSTRACT

The TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK) is known to be expressed on antigen-presenting cells and function as oncogenic drivers and as inhibitors of inflammatory responses. Both human and mouse CD8+ T cells are thought to be negative for TAM receptor expression. In this study, we show that T-cell receptor (TCR)-activated human primary CD8+ T cells expressed MERTK and the ligand PROS1 from day 2 postactivation. PROS1-mediated MERTK signaling served as a late costimulatory signal, increasing proliferation and secretion of effector and memory-associated cytokines. Knockdown and inhibition studies confirmed that this costimulatory effect was mediated through MERTK. Transcriptomic and metabolic analyses of PROS1-blocked CD8+ T cells demonstrated a role of the PROS1-MERTK axis in differentiation of memory CD8+ T cells. Finally, using tumor-infiltrating lymphocytes (TIL) from melanoma patients, we show that MERTK signaling on T cells improved TIL expansion and TIL-mediated autologous cancer cell killing. We conclude that MERTK serves as a late costimulatory signal for CD8+ T cells. Identification of this costimulatory function of MERTK on human CD8+ T cells suggests caution in the development of MERTK inhibitors for hematologic or solid cancer treatment.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , c-Mer Tyrosine Kinase/metabolism , Biomarkers , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cytokines/metabolism , Energy Metabolism , Gene Expression , Humans , Immunophenotyping , Lymphocyte Activation/immunology , Lymphocytes, Tumor-Infiltrating , Melanoma/genetics , Melanoma/immunology , Melanoma/metabolism , Melanoma/pathology , Protein S , Signal Transduction , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
13.
Melanoma Res ; 28(6): 510-520, 2018 12.
Article in English | MEDLINE | ID: mdl-30095598

ABSTRACT

The molecular properties of benign melanocytic lesions are poorly understood. Only a few studies have been carried out on specific nevi subtypes, including common nevocellular nevi (NCN) or Spitz nevi (SN). Genomic alterations in melanoma-associated oncogenes are typically absent in SN. In the present study, mRNA expressions of 25 SN and 15 NCN were analyzed. Molecular profiling was performed using the RNA NanoString nCounter Gene Expression Platform (number of genes=770). Marker discovery was performed with a training set consisting of seven SN and seven NCN samples from the same patients, and validation was performed using a second set consisting of 18 SN and eight NCN samples. Using the training set, 197 differentially expressed genes were identified in SN versus NCN. Of these, 74 genes were validated in the validation set (false discovery rate q≤0.13). In addition, using random forest and least absolute shrinkage and selection operator feature selection, a molecular signature of SN versus NCN was identified including 15 top-ranked genes. The present study identified a distinct molecular expression profile in SN compared with NCN, even when lesions were obtained from the same patients. Gene set analysis showed upregulation of gene pathways with increased expression of transcripts related to immunomodulatory, inflammatory, and extracellular matrix interactions as well as angiogenesis-associated processes in SN. These findings strongly indicate that SN represent a distinct group of melanocytic neoplasms and evolve differentially and not sequentially from NCN.


Subject(s)
Nevus, Epithelioid and Spindle Cell/diagnosis , RNA/metabolism , Skin Neoplasms/diagnosis , Adolescent , Adult , Female , Humans , Male , Middle Aged , Nevus, Epithelioid and Spindle Cell/pathology , Skin Neoplasms/pathology , Young Adult
14.
Clin Cancer Res ; 24(23): 5873-5882, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30061360

ABSTRACT

PURPOSE: Merkel cell carcinoma (MCC) is an aggressive skin cancer with neuroendocrine differentiation. There is an unmet need for MCC-specific blood-based surrogate biomarkers of tumor burden; circulating cell-free miRNA may serve this purpose. EXPERIMENTAL DESIGN: Expression of miR-375 was quantified in 24 MCC and 23 non-MCC cell lines, 67 MCC and 58 non-MCC tumor tissues, sera of 2 preclinical MCC models, and sera of 109 patients with MCC and 30 healthy controls by nCounter human-v2-miRNA expression or miR-375-specific real-time PCR assays. The patients' sera consisted of two retrospective (discovery and training) and two prospective (validation) cohorts. RESULTS: miR-375 expression was high in MCC cell lines and tissues compared with non-MCCs. It was readily detected in MCC-conditioned medium and sera of preclinical models bearing MCC xenografts. miR-375 levels were higher in sera from tumor-bearing patients with MCC than in tumor-free patients or healthy controls (P < 0.0005). Moreover, miR-375 serum levels correlated with tumor stage in tumor-bearing (P = 0.037) but not in tumor-free (P = 0.372) patients with MCC. miR-375 serum level showed high diagnostic accuracy to discriminate tumor-bearing and tumor-free patients with MCC as demonstrated by ROC curve analysis in the retrospective cohorts (AUC = 0.954 and 0.800) as well as in the prospective cohorts (AUC = 0.929 and 0.959). miR-375 serum level reflected dynamic changes in tumor burden of patients with MCC during therapeutic interventions. CONCLUSIONS: Circulating cell-free miR-375 proved as a surrogate marker for tumor burden in MCC without restriction to polyomavirus positivity; it thus appears to be useful for therapy monitoring and the follow-up of patients with MCC.


Subject(s)
Biomarkers, Tumor , Carcinoma, Merkel Cell/blood , Carcinoma, Merkel Cell/genetics , Circulating MicroRNA , MicroRNAs/genetics , Animals , Carcinoma, Merkel Cell/diagnosis , Case-Control Studies , Cell Line, Tumor , Chick Embryo , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Positron Emission Tomography Computed Tomography , Prognosis , ROC Curve , Real-Time Polymerase Chain Reaction , Tumor Burden , Xenograft Model Antitumor Assays
15.
Semin Immunopathol ; 39(3): 255-268, 2017 04.
Article in English | MEDLINE | ID: mdl-28074285

ABSTRACT

Characterizing the interaction of cancer cells with the host adaptive immune system is critical for understanding tumor immunology and the modus operandi of immunotherapeutic interventions to treat cancer. As the key cellular effectors of adaptive immunity, T cells are endowed with specialized receptors (the T cell receptor; TCR), to recognize and to eliminate cancer cells. The diversity of the TCR repertoire results from specialized genetic diversification mechanisms that generate an incredible variability allowing recognizing extensive collections of antigens. Based on the attainment and function of the TCR, the TCR repertoire is a mirror of the human immune response, and the dynamic changes of its usage can be assumed as a promising biomarker to monitor immunomodulatory therapies. Recent advances in multiplexed PCR amplification and massive parallel sequencing technologies have facilitated the characterization of TCR repertoires at high resolution even when only biomaterial of limited quantity and quality, such as formalin-fixed paraffin-embedded (FFPE) archived tissues, is available. Here, we review the concept framework and current experimental approaches to characterize the TCR repertoire usage in cancer including inherent technical and biological challenges.


Subject(s)
Neoplasms/immunology , Neoplasms/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Adaptive Immunity , Animals , Antigens, Neoplasm/immunology , Biomarkers , Biomarkers, Tumor , Clonal Evolution , Gene Rearrangement, T-Lymphocyte , High-Throughput Nucleotide Sequencing , Humans , Immunologic Surveillance , Immunotherapy , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Treatment Outcome
16.
J Cancer Res Clin Oncol ; 143(8): 1489-1497, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28405827

ABSTRACT

PURPOSE: Expression of O6-methylguanine-DNA methyltransferase (MGMT) in Merkel cell carcinoma (MCC) is very variable; thus, we tested whether this may be due to differential methylation of the MGMT gene promoter. METHODS: Quantitative analysis of MGMT mRNA and protein expression, as well as MGMT promoter methylation status, was performed in a series of tissue samples of MCC tumors, representing both primary and metastatic lesions, as well as in six MCC cell lines. RESULTS: These analyses revealed a very heterogeneous MGMT mRNA and protein expression in MCC both in vivo and in vitro. However, neither the MGMT mRNA nor protein expression correlated with the sensitivity of MCC cell lines toward the alkylating agent dacarbazine in vitro. Notably, increased methylation at the promoter of the MGMT gene was observed in 2/6 (33%) of the MCC cell lines; however, MGMT promoter methylation was absent in all MCC tissue samples. According to our results, albeit aberrant methylation of MGMT gene promoter can be observed in in vitro propagated MCC cell lines, it seems to be absent or very rare in MCC lesions in situ. CONCLUSION: Thus, the evaluation of this marker has no or only little significance for predicting response to therapy or for improving efficacy of demethylating agents in the treatment of MCC. Microenvironmental factors may play a role in explaining the different results between MCC cell lines and MCC samples.


Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Merkel Cell/genetics , DNA Methylation/genetics , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Tumor Suppressor Proteins/genetics , Animals , Biomarkers, Tumor/biosynthesis , Carcinoma, Merkel Cell/pathology , Cell Line, Tumor , DNA Modification Methylases/biosynthesis , DNA Repair Enzymes/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Mice , Promoter Regions, Genetic , Tumor Suppressor Proteins/biosynthesis , Xenograft Model Antitumor Assays
17.
J Cancer Res Clin Oncol ; 143(4): 613-617, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27990595

ABSTRACT

BACKGROUND: TERT promoter mutations were detected at high frequencies in several cancer types including melanoma. Previous reports showed that these recurrent mutations increase TERT gene expression and the use of TERT mutation status as prognostic factor was suggested. OBJECTIVES: Here we screen a panel of 115 melanoma tumor samples from Austrian patients to evaluate the prevalence and distribution of TERT promoter mutations. The association with clinical and tumor characteristics and the effect on overall survival was analyzed. METHODS: Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis. RESULTS: We identified TERT promoter mutations in 63 of 115 (54.8%) tumor samples. No statistical significant difference in mutation frequency between primary (22/40 [55%]) and metastatic lesions (41/75 [54.7%]) was detected. BRAF-/NRAS-mutated tumors showed a higher frequency of TERT mutations (pT OR 2.24, 95% CI 0.56-9.02, p = 0.3) (met OR 2.74, 95% CI 0.98-7.66, p = 0.05). In primary melanoma, the presence of alterations in TERT was associated with the carrier status of a common single-nucleotide polymorphism rs2853669 (OR 4.55, CI 1.18-17.52, p = 0.03). In this patient cohort, TERT promoter mutations were not associated with clinical characteristics such as the presence of ulceration or Breslow thickness or showed an effect on overall survival. CONCLUSION: Alterations in the TERT promoter region are one of the most frequent mutations in melanoma. Based on this analysis and preliminary evidence, prospective studies will be needed to evaluate the reliability of TERT promoter mutations as prognostic factors in melanoma.


Subject(s)
Melanoma/genetics , Neoplasm Metastasis/genetics , Promoter Regions, Genetic , Telomerase/genetics , Austria , Female , Humans , Male , Melanoma/pathology , Middle Aged
18.
Sci Rep ; 7(1): 2290, 2017 05 23.
Article in English | MEDLINE | ID: mdl-28536458

ABSTRACT

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.


Subject(s)
Antigen Presentation/immunology , Carcinoma, Merkel Cell/immunology , Epigenesis, Genetic/immunology , Histocompatibility Antigens Class I/immunology , Skin Neoplasms/immunology , Animals , Antigen Presentation/genetics , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/metabolism , Cell Line, Tumor , Epigenesis, Genetic/genetics , Female , Gene Expression Regulation, Neoplastic/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Immunohistochemistry , Mice, Inbred NOD , Mice, SCID , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Transplantation, Heterologous
19.
J Cancer Res Clin Oncol ; 143(7): 1199-1207, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28314930

ABSTRACT

BACKGROUND: Recent advances in sequencing technologies supported the development of molecularly targeted therapy in cancer patients. Thus, genomic analyses are becoming a routine part in clinical practice and accurate detection of actionable mutations is essential to assist diagnosis and therapy choice. However, this is often challenging due to major problems associated with DNA from formalin-fixed paraffin-embedded tissue which is usually the primary source for genetic testing. OBJECTIVES: Here we want to share our experience regarding major problems associated with FFPE DNA used for PCR-based sequencing as illustrated by the mutational analysis of ERBB4 in melanoma. We want to focus on two major problems including extensive DNA fragmentation and hydrolytic deamination as source of non-reproducible sequence artifacts. Further, we provide potential explanations and possible strategies to minimize these difficulties and improve the detection of targetable mutations. METHODS: Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis. RESULTS: Analysis of Sanger sequencing data revealed a total of 46 ERBB4 mutations in 27 of 96 samples including the identification of 11 mutations at three previously unknown mutational hotspots. Unfortunately, we were not able to confirm any assumed hotspot mutation within repeated sequencing of relevant amplicons suggesting the detection of sequence artifacts most likely caused by DNA lesions associated with FFPE tissues. CONCLUSION: Since DNA from FFPE tissue is usually the primary source for mutational analyses, appropriate measures must be implemented in the workflow to assess DNA damage in formalin-fixed tissue to ensure accurate detection of actionable mutations and minimize the occurrence of sequence artifacts.


Subject(s)
Artifacts , DNA Mutational Analysis/methods , Paraffin Embedding , Receptor, ErbB-4/genetics , Tissue Fixation , Base Sequence , DNA/analysis , Formaldehyde , Humans , Melanoma/genetics , Polymerase Chain Reaction/methods , Receptor, ErbB-4/analysis
20.
Sci Rep ; 6: 21678, 2016 Feb 23.
Article in English | MEDLINE | ID: mdl-26902929

ABSTRACT

Merkel cell carcinoma (MCC) is a virally associated cancer characterized by its aggressive behavior and strong immunogenicity. Both viral infection and malignant transformation induce expression of MHC class I chain-related protein (MIC) A and B, which signal stress to cells of the immune system via Natural Killer group 2D (NKG2D) resulting in elimination of target cells. However, despite transformation and the continued presence of virally-encoded proteins, MICs are only expressed in a minority of MCC tumors in situ and are completely absent on MCC cell lines in vitro. This lack of MIC expression was due to epigenetic silencing via MIC promoter hypo-acetylation; indeed, MIC expression was re-induced by pharmacological inhibition of histone deacetylases (HDACs) both in vitro and in vivo. This re-induction of MICs rendered MCC cells more sensitive to immune-mediated lysis. Thus, epigenetic silencing of MICs is an important immune escape mechanism of MCCs.


Subject(s)
Carcinoma, Merkel Cell/genetics , Gene Silencing/immunology , Histocompatibility Antigens Class I/genetics , Histone Deacetylases/genetics , Killer Cells, Lymphokine-Activated/immunology , Skin Neoplasms/genetics , Acetylation/drug effects , Animals , Carcinoma, Merkel Cell/drug therapy , Carcinoma, Merkel Cell/immunology , Carcinoma, Merkel Cell/pathology , Cell Line, Tumor , Cytotoxicity, Immunologic , Gene Silencing/drug effects , Histocompatibility Antigens Class I/immunology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/immunology , Histones/genetics , Histones/immunology , Humans , Hydroxamic Acids/pharmacology , Killer Cells, Lymphokine-Activated/cytology , Killer Cells, Lymphokine-Activated/drug effects , Mice , Mice, Inbred NOD , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/immunology , Plicamycin/analogs & derivatives , Plicamycin/pharmacology , Promoter Regions, Genetic/drug effects , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Vorinostat , Xenograft Model Antitumor Assays
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